Synthetic oligonucleotides are widely used compounds in disparate fields such as molecular biology and DNA-based diagnostics and therapeutics.
General Considerations
To be useful in the extensive range of the different applications outlined above, oligonucleotides have to satisfy a large number of different requirements. As therapeutics, for instance, a useful oligonucleotide must be able to penetrate the cell membrane, have good resistance to extra- and intracellular nucleases and preferably have the ability to recruit endogenous enzymes like RNAseH. In DNA-based diagnostics and molecular biology, other properties are important such as, for instance, the ability of oligonucleotides to act as efficient substrates for a wide range of different enzymes evolved to act on natural nucleic acids, such as e.g. polymerases, kinases, ligases and phosphatases. The fundamental property of oligonucleotides, however, which underlies all uses is their ability to recognise and hybridise sequence specifically to complementary single stranded nucleic acids employing either Watson-Crick hydrogen bonding (A-T and G-C) or other hydrogen bonding schemes such as the Hoogsteen mode. There are two important terms, affinity and specificity, commonly used to characterise the hybridisation properties of a given oligonucleotide. Affinity is a measure of the binding strength of the oligonucleotide to its complementary target sequence (expressed as the thermostability (Tm) of the duplex). Each nucleobase pair in the duplex adds to the thermostability and thus affinity increases with increasing size (number of nucleobases) of the oligonucleotide. Specificity is a measure of the ability of the oligonucleotide to discriminate between a fully complementary and a mismatched target sequence. In other words, specificity is a measure of the loss of affinity associated with mismatched nucleobase pairs in the target.
At constant oligonucleotide size, the specificity increases with increasing number of mismatches between the oligonucleotide and its targets (i.e. the percentage of mismatches increases). Conversely, specificity decreases when the size of the oligonucleotide is increased at a constant number of mismatches (i e. the percentage of mismatches decreases). Stated another way, an increase in the affinity of an oligonucleotide occurs at the expense of specificity and vice-versa.
Given the shortcomings of natural oligonucleotides, new approaches for enhancing specificity and affinity are highly desirable for DNA-based therapeutics, diagnostics and for molecular biology techniques in general.
Conformationally Restricted Nucleosides
It is known that oligonucleotides undergo a conformational transition in the course of hybridising to a target sequence, from the relatively random coil structure of the single stranded state to the ordered structure of the duplex state.
Thus, conformational restriction has in recent years been applied to oligonucleotides in the search for analogues displaying improved hybridisation properties compared to the unmodified (2′-deoxy)oligonucleotides. For example bicyclo[3.3.0]nucleosides with an additional C-3′,C-5′-ethano-bridge (M. Tarköy, M. Bolli, B. Schweizer and C. Leumann, Helv. Chem. Acta, 1993, 76, 481; Tarköy and C. Leumann, Angew. Chem., Int. Ed. Engl., 1993, 32, 1432; M. Egli, P. Lubini, M. Dobler and C. Leumann, J. Am. Chem. Soc., 1993, 115, 5855; M. Tarköy, M. Bolli and C. Leumann, Helv. Chem. Acta, 1994, 77, 716; M. Bolli and C. Leumann, Angew. Chem., Int. Ed. Engl., 1995, 34, 694; M. Bolli, P. Lubini and C. Leumann, Helv. Chem. Acta, 1995, 78, 2077; J. C. Litten, C. Epple and C. Leumann, Bioorg. Med. Chem. Lett., 1995, 5, 1231; J. C. Litten and C. Leumann, Helv. Chem. Acta, 1996, 79, 1129; M. Bolli, J. C. Litten, R. Schültz and C. Leumann, Chem. Biol., 1996, 3, 197, M. Bolli, H. U. Trafelet and C. Leumann, Nucleic Acids Res., 1996, 24, 4660), bicarbocyclo[3.1.0]nucleosides with an additional C-1′,C6′- or C6′,C4′-methano-bridge (K.-H. Altmann, R. Kesselring, E. Francotte and G. Rihs, Tetrahedron Lett., 1994, 35, 2331; K.-H. Altmann, R. Imwinkelned, R. Kesselring and G. Rihs, Tetrahedron Lett., 1994, 35, 7625; V. E. Marquez, M. A. Siddiqui, A. Ezzitouni, P. Russ, J. Wang, R. W. Wagner and M. D. Matteucci, J. Med. Chem., 1996, 39, 3739; A. Ezzitouni and V. E. Marquez, J. Chem. Soc., Perkin Trans. 1, 1997, 1073), bicyclo[3.3.0]- and [4.3.0] nucleoside containing an additional C-2′,C-3′-dioxalane ring synthesised as a dimer with an unmodified nucleoside where the additional ring is part of the internucleoside linkage replacing a natural phosphordiester linkage (R. J. Jones, S. Swaminathan, J. F. Milagan, S. Wadwani, B. S. Froehler and M. Matteucci, J. Am. Chem. Soc., 1993, 115, 9816; J. Wang and M. D. Matteucci, Bioorg. Med. Chem. Lett., 1997, 7, 229), dimers containing a bicyclo[3.1.0]nucleoside with a C-2′,C-3′-methano bridge as part of amide- and sulfonamide-type internucleoside linkages (C. G. Yannopoulus, W. Q. Zhou, P. Nower, D. Peoch, Y. S. Sanghvi and G. Just, Synlett, 1997, 378), bicyclo[3.3.0]glucose-derived nucleoside analogue incorporated in the middle of a trimer through formacetal internucleoside linkages (C. G. Yannopoulus. W. Q. Zhou, P. Nower, D. Peoch, Y. S. Sanghvi and G. Just, Synlett, 1997, 378) and bicyclic[4.3.0]- and [3.3.0]nucleosides with additional C-2′,C-3′-connected six- and five-membered ring (P. Nielsen, H. M. Pfundheller, J. Wengel, Chem. Commun., 1997, 826; P. Nielsen, H. M. Pfundheller, J. Wengel, XII International Roundtable: Nucleosides, Nucleotides and Their Biological Applications; La Jolla, Calif., Sep. 15–19, 1996; Poster PPI 43) have been synthesised and incorporated into oligodeoxynucleotides. Unfortunately, oligonucleotides comprising these analogues form in most cases less stable duplexes with complementary nucleic acids compared to the unmodified oligonucleotides. In cases where a moderate improvement in duplex stability is observed, this relates only to either a DNA or an RNA target, or it relates to fully but not partly modified oligonucleotides or vice versa.
An appraisal of most of the reported analogues are further complicated by the lack of data on analogues with G, A and C nucleobases and lack of data indicating the specificity and mode of hybridisation. In many cases, synthesis of the reported monomer analogues is very complex while in other cases the synthesis of fully modified oligonucleotides is incompatible with the widely used phosphoramidite chemistry standard.
Recently, oligomers comprising Locked Nucleic Acids (LNA) have been reported (Nielsen, P., Pfundheller, H. M., Olsen, C. E. and Wengel, J., J. Chem. Soc., Perkin Trans. 1, 1997, 3423; Nielsen, P., Pfundheller, H. M., Wengel, J., Chem. Commun., 1997, 9, 825, Christensen, N. K., Petersen, M., Nielsen, P., Jacobsen, J. P. and Wengel, J., J. Am. Chem. Soc., 1998, 120, 5458; Koshkin, A. A. and Wengel, J., J. Org. Chem., 1998, 63, 2778; Obika, S., Morio, K.-I., Hari, Y. and Imanishi, T., Bioorg. Med. Chem. Lett., 1999, 515). Interestingly, incorporation of LNA monomers containing a 2′-O,4′-C-methylene bridge into an oligonucleotide sequence led to unprecedented improvement in the hybridisation ability of the modified oligonucleotide (Singh, S. K., Nielsen, P., Koshkin, A. A., Olsen, C. E. and Wengel, J., Chem. Commun., 1998, 455; Koshkin, A. K., Singh, S. K., Nielsen, P., Rajwanshi, V. K., Kumar, R., Meldgaard, M., Olsen, C. E., and Wengel, J., Tetrahedron, 1998, 54, 3607; Koshkin, A. A. Rajwanshi, V. K., and Wengel, J., Tetrahedron Lett., 1998, 39, 4381; Singh, Sanjay K. and Wengel, J., Chem. Commun, 1998, 1247; Kumar, R., Singh, S. K, Koshkin, A. A., Rajwanshi, V. K., Meldgaard, M., and Wengel. J., Bioorg. Med. Chem. Lett., 1998, 8, 2219; Obika, S. et al. Tetrahedron Lett., 1997, 38, 8735; Obika, S. et al. Tetrahedron Left., 1998, 39, 5401, Singh, S. K., Kumar, R., and Wengel, J., J. Org. Chem., 1998, 63, 6078; Koshkin, A. A., Nielsen, P., Meldgaard, M., Rajwanski, V. K., Singh, S. K., and Wengel, J., J. Am. Chem. Soc., 1998, 120, 13252; Singh, S. K., Kumar, R., and Wengel, J., J. Org. Chem., 1998, 63, 10035). Oligonucleotides comprising these LNA monomers and the corresponding 2′-thio-LNA analogue form duplexes with complementary DNA and RNA with thermal stabilities not previously observed for bi- or tricyclic nucleosides modified oligonucleotides (ΔTm/modification=+3 to +11° C.) and show improved selectivity.
In a series of papers, Seela et al. have studied xylo-DNA (FIG. 1, Base=adenin-9-yl, cytosin-1-yl, guanin-9-yl or thymin-1-yl) comprising one or more 2′-deoxy-β-D-xylofuranosyl nucleotide monomers (Rosemeyer, H.; Seela, F. Helv. Chem. Acta 1991, 74, 748; Rosemeyer, H.; Krecmerova, M.; Seela, F. Helv. Chem. Acta 1991, 74, 2054; Seela, F.; Wörner, Rosemeyer, H. Helv. Chem. Acta 1994, 77, 883; Seela, F.; Heckel, M.; Rosemeyer, H. Helv. Chem. Acta 1996, 79, 1451; Rosemeyer, H.; Seela, F. Nucleosides Nucleotides, 1995, 14, 1041; Schoeppe, A.; Hinz, H.-J., Rosemeyer, H.; Seela, F. Eur. J. Biochem. 1996, 239, 33). Compared with the corresponding natural 2′-deoxy-β-D-ribofuranosyl oligonucleotides, xylo-DNA, generally, display a mirror-image-like secondary structure, entropically favourable duplex formation, increased stability towards exonucleases, and, for oligonucleotides comprising a small number of 2′-deoxy-β-D-xylofuranosyl monomers, decreased thermal affinity towards complementary DNA (Rosemeyer, H.; Seela, F. Helv. Chem. Acta 1991, 74, 748; Rosemeyer, H.; Krecmerova, M.; Seela, F. Helv. Chem. Acta 1991, 74, 2054; Seela, F.; Wörner, Rosemeyer, H. Helv. Chem. Acta 1994, 77, 883; Seela, F.; Heckel, M.; Rosemeyer, H. Helv. Chem. Acta 1996, 79, 1451).